Fig 2: The small molecule inhibitor, GSK715, blocks H3R17me2a protein level after dual administration in MC3T3 cells. (A) MC3T3 cells were treated by GSK715 (Type 1 PRMT inhibitor) on day 0 and day 2 (2× treatment), and protein lysates were collected on day 3. (B) Western blotting shows PRMT1 and PRMT4/CARM1 protein levels do not change by dual administration of either 2 µM or 5 µM of GSK715 on day 3 compared to the GAPDH protein level. (C) Western blot analysis shows that GSK715 reduces levels of H3R17me2a (mediated by Pmrt1) but not H4R3me2a (which is mediated by PRMT4/CARM1). Administration of GSK715 at two different doses (2 µM and 5 µ) has no biological effects on total H4 or H3 protein levels (representative gel of 3 biological replicates).

Fig 3: Loss of Prmt1, Prmt4/Carm1, and Prmt5 upon siRNA transfection has different effects on calcium deposition of MC3T3 cells. (A) A time course of osteoblast differentiation shows upregulation of (B) representative Alizarin Red stained cultures. The results show that MC3T3 cell mineralization is enhanced upon siPrmt1 treatment, remains the same as control upon siCarm1 treatment, and is decreased after siPrmt5 knock-down. (C) Quantification of mineralization in differentiating cells on day 30 using Image J software. Statistical significance was determined by two-way ANOVA (n = 3, mean + STD; * = p < 0.05 and **** = p < 0.0001). We note that for each of the siRNAs, we stained the cultures as pairs of nsRNA vs siRNA at different times when mineralization would be apparent in either the control or treatment group; hence, mineralization in controls differs for each of nsRNA/siRNA pairs (respectively, for PRMT1, CARM1 and PRMT5). The time course endpoint of Alizarin Red staining is determined by the appearance of visual evidence for nodular calcium deposition and varies between experiments (day 21 to day 32).

Fig 4: PRMT loss of function validation by RT-qPCR and western blotting in differentiating MC3T3 cells. RNAs and proteins were isolated on day 3 to validate Prmt knock-down by RT-qPCR and western blotting. (A) Parallel RT-qPCR analysis for Prmt mRNAs (Prmt1, Prmt4/Carm1, Prmt5) on day 3 of differentiation (n = 3, mean ± STD; ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001). (B) Western blotting analysis for PRMT1, PRMT4/CARM1, PRMT5 and GAPDH was performed on day 3 of differentiation.

Fig 5: PRMT knock-down effects on mRNA levels of other PRMT members during osteoblast differentiation. Time course expression analysis using RT-qPCR shows that siRNA depletion since day 0 suppresses select Prmt mRNA levels. (A)Prmt4/Carm1 mRNA levels are enhanced upon Prmt1 knock-down at day 6 after induction of osteoblast differentiation. (B)Prmt1 and Prmt5 mRNA levels do not show dramatic change after Prmt4/Carm1 loss of function. (C)Prmt1 and Prmt4/Carm1 mRNA levels do not show appreciable modulations in mRNA levels upon PRMT5 loss of function over the osteoblast differentiation in 15 days. (n = 3, mean ± STD; ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001).